1. Field of the Invention
The present invention relates generally to an apparatus and process for conducting electrophoresis and transfer and more particularly to an apparatus and process for automatically conducting electrophoresis and transfer whereby the handling of the gel is substantially reduced.
2. Description of the Prior Art
As is well known in the art, various problems and difficulties have been encountered in conducting electrophoresis and transfer of macromolecules such as nucleic acid DNA/RNA fragments. A popular electrophoresis and transfer procedure is commonly referred to as the Southern Blot Technique.
Basically, the electrophoresis procedure provides for the migration of charged macromolecules through a suitable retarding medium under the influence of a horizontal electro-transfer function (i.e., an electric field) During the horizontal transfer function, the macromolecules of high molecular weight migrate at a slower rate through the medium than do the macromolecules of lower molecular weight. The normal retarding medium comprises a conventional electrophoresis gel that is supported within a tray. Several wells are formed transversely across the gel, the wells being adapted to receive individual DNA, RNA or protein samples for processing. The samples are positioned so as to be subjected to the electrophoresis process, at which time the tray is at least partially filled with an electrolyte buffer. Electrodes are commonly positioned at each end of the gel tray. A sufficient voltage difference is applied to the electrodes to cause the macromolecules of the sample to migrate from the wells longitudinally along the gel, so that they are separated according to their molecular weight.
At this point, the gel is generally removed from its tray. Macromolecules such as DNA/RNA fragments are then generally depurinated i.e., broken into smaller fragments and subsequently denatured i.e., separated into single strands for subsequent transfer onto a suitable membrane.
There are many occasions in the steps of conventional electrophoresis, depurination, denaturation and transfer of the macromolecules to the membrane that cause difficulties which allow for errors as well as loss of considerable time and money. Various devices have been provided previously for carrying out electrophoresis such as can be found in U.S. Pat. No. 4,415,418.
In U.S. Pat. No. 4,726,889 to J.D. Love et al there is disclosed an apparatus for carrying out horizontal gel electrophoresis for separation and subsequent vacuum-assisted transportation of macromolecules to a support membrane to facilitate detection The entire procedure is conducted in one cartridge.
U.S. Pat. No. 4,911,816 also to J.D. Love et al discloses an apparatus to very similar to that of the above noted patent to J.D. Love et al. This application is a continuation of application Ser. No. 077,240, now abandoned, which is a continuation-in-part of U.S. Pat. No. 4,726,889 and U.S. Pat. No. 4,756,809.
The above referenced patents do not allow for a fully automatic procedure. To allow for a fully automatic electrophoresis operation having high-quality results, different and additional sophisticated features are necessary. In order to achieve a truly automatic apparatus for Southern blotting (an apparatus that will operate all by itself after loading the gel until the completion of the transfer) it is necessary to find ways to prevent any liquid from penetrating into the binding membrane under the gel and/or into the mechanical support platform that has to previous for liquid to allow suction blotting.
There are several independent reasons for this requirement:
1. During electrophoresis, the electrolyte buffer will completely or at least partially penetrate into the space under the gel. This is turn will cause deviations of the electric field lines that will follow the nonlinear boundaries of the lower liquid surface. As a result of this the macromolecule (e.g., DNA) bands of interest will at least partially exit the gel on its lower surface, thereby binding them in wrong places to any accessible membrane positioned under the gel. The result will often be a messy, unreliable image of the band patterns that are to be transferred later in the process.
Another problem is inhomogeneity of field strength due to said field distortions, leading to deviations and errors in migration distances.
In the actual operation of the apparatus disclosed in the J.D. Love et al patents there is no continuous automatic operation provided because the DNA binding membrane cannot be present during the electrophoresis and chemical treatments for the reasons discussed above. This membrane is manually positioned under the gel after all the chemical treatments of the gel are completed.
2. Another reason why the membrane needs to be separated from the gel is that, during the depurination and denaturation steps, strong chemicals would contact the membrane and alter its later binding performance in ill defined ways, if not completely destroying its binding characteristics.
Accordingly, after reviewing the following disclosure it will be readily understood that the present invention provides various unique operating structures and processing features which make it possible to construct a truly and fully automatic electrophoresis, chemical treatment and transfer apparatus as heretofore not found in the art.